CN112903641A - Biosensor for detecting histone modification enzyme and detection method and application thereof - Google Patents
Biosensor for detecting histone modification enzyme and detection method and application thereof Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N2021/6432—Quenching
Abstract
The invention provides a biosensor for detecting histone modification enzyme, a detection method and application thereof, belonging to the technical field of detection and analysis. The biosensor at least comprises a histone modification enzyme enzymatic substrate, a DNA probe, a magnetic bead and a rolling circle amplification template; wherein the histone modification enzyme enzymatic substrate is carboxyl-terminal biotinylated polypeptide; the DNA probe comprises an aptamer and a primer sequence; the aptamer recognizes a histone modification enzyme to be detected; the primer is combined and complemented with the rolling circle amplification template. The invention is based on aptamer-mediated histone modification site specific rolling circle amplification, so that the unmarked detection is carried out on the histone modification enzyme on the femtomolar level without any antibody and radioactive/fluorescent mark, and the introduction of the histone modification site specific rolling circle amplification can greatly improve the detection sensitivity, thereby having good practical application value.
Description
Technical Field
The invention belongs to the technical field of detection and analysis, and particularly relates to a biosensor for detecting histone modification enzyme, and a detection method and application thereof.
Background
The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.
Histones are key protein components of chromatin and are responsible for packaging genomic DNA in eukaryotic cells. In the nucleus, four core histones H2A, H2B, H3 and H4 are wrapped by 147bp DNA fragments to form nucleosome structures, and the repeated nucleosomes are further condensed into chromatin with the aid of the linker histone H1. Proteins are highly conserved evolutionarily, and their N-termini are often subjected to post-translational modifications catalyzed by various histone modification enzymes (e.g., acetylation, methylation, phosphorylation, ubiquitination, and the like). These normal histone modifications play a key role in basic cellular functions, including transcriptional regulation, messenger RNA splicing, and memory formation. However, dysregulation of histone modifying enzymes may interfere with histone modification and affect downstream biological functions.
Heretofore, radioactive assays based on radioisotope-labeled substrates have been widely used for histone modification enzyme assays, but the introduction of hazardous substances and heterogeneous procedures have limited their practical application. In addition, enzyme-linked assays have been introduced to perform nonradioactive detection of histone modifying enzymes in a homogeneous manner, but they require the cooperation of multiple enzymes, which is costly and complex. Mass spectrometry can detect histone modifying enzymes by directly identifying modified substrate residues, but it involves expensive instruments and complicated procedures. In recent years, a variety of antibody-based histone modification enzyme detection methods have been developed, combining quantum dot-based fluorescence resonance energy transfer, gold nanoparticle-based colorimetric detection, and total internal reflection fluorescence-based single molecule detection. Antibodies are relatively expensive and difficult to produce from cells and animals, and are poorly stable under harsh assay conditions. To date, sensitive detection of low abundance histone modification enzymes remains challenging because the enzyme/protein signal cannot be amplified like nucleic acids.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a biosensor for detecting histone modification enzyme and a detection method and application thereof. The present invention is based on aptamer-mediated histone modification site-specific rolling circle amplification for label-free detection of histone modification enzymes at femtomolar levels, aptamer-primers can be combined with templates for rolling circle amplification to produce abundant single-stranded rolling circle amplification products, which can be measured simply in a label-free manner by using SYBR Gold dye. The assay uses an aptamer for modification site recognition, uses SYBR Gold dye for signal measurement, has the detection limit as low as 200 femtomoles per liter, does not need to relate to any antibody and radioactive/fluorescent labels, and introduces histone modification site specific rolling circle amplification to greatly improve the detection sensitivity, thereby having good practical application value.
In order to achieve the technical purpose, the technical scheme adopted by the invention is as follows:
in a first aspect of the invention, a biosensor for detecting a histone modification enzyme is provided, the biosensor comprising at least a histone modification enzyme enzymatic substrate, a DNA probe, a magnetic bead and a rolling circle amplification template;
wherein the content of the first and second substances,
the histone modification enzyme enzymatic substrate is carboxyl-terminal biotinylated polypeptide;
the DNA probe comprises an aptamer and a primer sequence;
the aptamer can effectively identify histone modification enzyme to be detected;
the primer and the rolling circle amplification template can be combined and complemented.
Further, a spacer is provided between the aptamer and primer sequence to reduce steric hindrance in the aptamer-peptide binding and primer-template base pairing.
The magnetic beads are streptavidin-coated magnetic beads, under the condition that histone modification enzyme to be detected exists, the histone modification enzyme catalyzes a histone modification enzyme enzymatic substrate to generate modification sites, and due to high binding affinity between the modification sites and an aptamer, the obtained substrate can be assembled on the streptavidin-coated magnetic beads through specific biotin-streptavidin interaction, so that a magnetic bead-peptide chain-aptamer-primer composite structure is formed.
Further, the biosensor may further include a fluorescent dye, which may be a SYBR Gold dye, and the SYBR Gold dye may bind to the nucleic acid to induce more than 1000-fold signal enhancement due to high quantum yield of the SYBR Gold dye-nucleic acid complex, thereby performing signal measurement.
When the histone modifying enzyme is acetyltransferase Tip60, the histone modifying enzyme enzymatic substrate is a H4 polypeptide substrate, the amino acid sequence of which is as follows: GGKGLGKGGAKRHRK-biotin (SEQ ID NO. 1);
the nucleotide sequence of the DNA probe is as follows:
the nucleotide sequence of the rolling circle amplification template is as follows:
CTC AGC TGT GTA ACAACA TGA AGA TTG TAG GTC AGA ACT CAC CTG TTAGAA ACT GTG AAG ATC GCT TAT TAT GTC CTA TC(SEQ ID NO.3)；
the underlined bases indicate complementary regions where the primer sequence of the DNA probe binds to the RCA template, and the bases in the shaded area indicate the aptamer sequence of the DNA probe.
In a second aspect of the invention, there is provided a method of detecting a histone modification enzyme, the method comprising:
1) incubating the substance to be tested and a histone modification enzyme enzymatic substrate together, and adding a DNA probe for continuous incubation;
2) adding magnetic beads into the step 1) for incubation, washing the magnetic beads after the incubation is finished, dissociating the rest DNA probes, and then continuing high-temperature incubation treatment;
3) adding a rolling circle amplification template into the DNA probe eluted in the step 2) for rolling circle amplification.
The method for detecting the histone modification enzyme also comprises the step of carrying out fluorescent staining on the rolling circle amplification product in the step 3), thereby realizing real-time fluorescent detection and/or gel electrophoresis and fluorescent spectrum measurement.
In a third aspect of the present invention, there is provided the use of the above-mentioned biosensor and/or detection method in the detection of histone modification enzyme activity and/or screening of a drug related to histone modification enzyme.
Although the present invention provides a relevant detection biosensor and detection method by taking the detection of histone modification enzymes as an example, it is obvious that the substitution of substrate peptide chains and DNA probe sequences for detecting other proteases or other enzymes based on the concept of the present invention is also conceivable, and thus the present invention shall also fall within the protection scope of the present invention.
The beneficial technical effects of the technical scheme are as follows:
1. the histone modification specificity DNA aptamer is firstly used for measuring the activity of the histone modification enzyme, and a new way is opened for the amplification detection of the histone modification enzyme by integrating various DNA amplification technologies (such as polymerase chain reaction, exponential amplification reaction, strand displacement amplification and loop-mediated isothermal amplification). The method is label-free, SYBR Gold fluorescence is used as signal output, and the participation of expensive fluorescent labels and false positive caused by incomplete fluorescence quenching are avoided. Aptamers replace expensive, poorly stable antibodies, thereby enabling specific transduction of enzyme signals to amplifiable DNA signals.
2. The sensitivity is high: the limit of detection of this detection method is as low as 200 femtomoles per liter, which is the most sensitive histone modification enzyme detection method reported to date. Importantly, the detection method can be easily extended to the detection of other histone modification enzymes by simply changing peptide and DNA sequences, and provides a powerful platform for biomedical research and clinical diagnosis related to the histone modification enzymes.
3. The specificity is good: the used Tip60 specific substrate peptide chain and specific aptamer enable the technical scheme to have higher detection specificity; in addition, each reaction condition in the technical scheme is also carefully optimized, so that the detection specificity is excellent.
4. The application range is wide: the technical scheme can realize quantitative detection of histone acetyltransferase Tip60 activity and can well carry out experiments on Tip60 inhibitor screening; in addition, the technical scheme can also be used for the dynamic correlation determination of Tip60 and the sensitive determination of the enzyme activity in a practical sample (cancer cell) with complex components, and has wide application range. Specific aptamers of other histone modification enzymes are screened by an exponential enrichment system ligand evolution technology, and an amplification technology is combined, so that the specific and ultrasensitive detection of the histone modification enzymes is realized. Therefore, the technical scheme has wide application range.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.
FIG. 1: schematic diagram of label-free aptamer sensor constructed for ultrasensitive detection of Tip60 based on histone modification site-specific rolling circle amplification. The insert shows the structure of the DNA probe.
FIG. 2 is a graph showing the correlation between the products of rolling circle amplification reaction in lipogelanalysis and fluorescence emission spectra of SYBRgold dye, wherein A is the product of rolling circle amplification reaction in lipogelanalysis. And (3) M times: DL2000 DNA standard; lane 1: phi29+ DNA probe + rolling circle amplification template; lane 2: tip60+ DNA probe + rolling circle amplification template; lane 3: tip60+ phi29+ DNA probe; lane 4: tip60+ phi29+ rolling circle amplification template; lane 5: tip60+ phi29+ DNA probe rolling circle amplification template; b is the fluorescence emission spectrum of the SYBRgold dye. Polypeptide substrate + DNA probe (line 4), Tip60+ polypeptide substrate + DNA probe (line 3), polypeptide substrate + DNA probe + rolling circle amplification template (line 2) and Tip60+ polypeptide substrate + DNA probe + rolling circle amplification template (line 1). 200 nmol per liter Tip60, 60 micromol per liter polypeptide substrate, 0.6 Uuphi 29 DNA polymerase, 500 nmol per liter DNA probe and 0.4 nmol per liter rolling circle amplification template were used in the experiment.
FIG. 3 is a graph relating to the fluorescence response of Tip60, wherein A is the fluorescence emission spectrum in response to serial dilutions of Tip 60; b is a linear relationship between fluorescence intensity and the logarithm of Tip60 concentration. Error bars represent standard deviations from three independent measurements.
FIG. 4 shows the fluorescence intensity in response to BSA (10. mu.g per ml), Dam (10 units per ml), CpG (10 units per ml), PKA (10 units per ml), PNK (10 units per ml), TIP60(200 nmol per liter), and no protein/enzyme in the control. Error bars represent standard deviations from three independent measurements.
Fig. 5 is a graph of the relative activity of Tip60 on different concentrations of small organic molecule NU 9056. The inset shows the structure of small organic molecule NU 9056. Error bars represent standard deviations from three independent measurements.
FIG. 6 is a plot of fluorescence emission spectra correlation of SYBR Gold from cervical cancer cells; wherein A is the fluorescence emission spectrum of SYBR Gold in response to different numbers of cervical cancer cells; and B is the relative activity of Tip60 responding to 10000 cervical cancer cells treated by organic small molecule NU 9056. Error bars represent standard deviations from three independent measurements.
Detailed Description
It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise. It is to be understood that the scope of the invention is not to be limited to the specific embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention.
As mentioned above, the existing detection of enzymes such as histone modification enzyme generally has the defects of long time consumption, large sample consumption, complex and tedious preparation or detection process, poor sensitivity and the like.
In view of the above, it is necessary to develop a simple and sensitive method for detecting the activity of histone modification enzymes. The invention develops aptamer-mediated histone modification site-specific rolling circle amplification for the first time, and is used for label-free detection of histone modification enzymes at femtomolar level. Tip60 is an acetyltransferase that catalyzes the acetylation of lysine residues on H4 histones. In the presence of Tip60, modification sites are catalytically generated. Due to the high affinity between the modification site and the aptamer, a magnetic bead-peptide chain-aptamer-primer biological composite structure is formed. Aptamer-primers can bind to templates for rolling circle amplification, yielding abundant single-stranded rolling circle amplification products that can be measured simply in a label-free manner by using SYBR Gold dyes. This assay uses aptamers for modification site recognition and SYBR Gold dyes for signal measurement with detection limits as low as 200 femtomoles per liter, the most sensitive histone modification enzyme detection method reported to date. The technology does not need to relate to any antibody and radioactive/fluorescent label, and the detection sensitivity can be greatly improved by introducing histone modification site specific rolling circle amplification. The technology can be further applied to enzyme activity mechanical analysis, enzyme activity inhibitor screening and histone modification enzyme activity detection in different cell lines, and provides a powerful detection technology platform for biomedical research and clinical application research related to histone modification enzyme.
In one embodiment of the present invention, there is provided a biosensor for detecting a histone modification enzyme, the biosensor comprising at least a histone modification enzyme enzymatic substrate, a DNA probe, a magnetic bead and a rolling circle amplification template;
wherein the content of the first and second substances,
the histone modification enzyme enzymatic substrate is carboxyl-terminal biotinylated polypeptide;
the DNA probe comprises an aptamer and a primer sequence;
the aptamer can effectively identify histone modification enzyme to be detected;
the primer and the rolling circle amplification template can be combined and complemented.
In yet another embodiment of the invention, a spacer is provided between the aptamer and primer sequence to reduce steric hindrance in the aptamer-peptide binding and primer-template base pairing.
In another embodiment of the present invention, the magnetic beads are streptavidin-coated magnetic beads, and in the presence of a histone modification enzyme to be detected, the histone modification enzyme catalyzes a histone modification enzyme enzymatic substrate to generate a modification site, and due to high binding affinity between the modification site and an aptamer, the obtained substrate can be assembled on the streptavidin-coated magnetic beads through a specific biotin-streptavidin interaction, thereby forming a magnetic bead-peptide chain-aptamer-primer composite structure.
In yet another embodiment of the present invention, the biosensor further comprises a fluorescent dye, which may be a SYBR Gold dye, which may bind to a nucleic acid to induce more than 1000-fold signal enhancement due to the high quantum yield of the SYBR Gold dye-nucleic acid complex, thereby performing signal measurement.
In yet another embodiment of the invention, when the histone modifying enzyme is the acetyltransferase Tip60, the histone modifying enzyme enzymatic substrate is a H4 polypeptide substrate having the following amino acid sequence: GGKGLGKGGAKRHRK-biotin (SEQ ID NO. 1);
in another embodiment of the present invention, the DNA probe has the following nucleotide sequence:
in another embodiment of the present invention, the rolling circle amplification template has the following nucleotide sequence:
CTC AGC TGT GTA ACA ACATGA AGA TTG TAG GTC AGA ACT CAC CTG TTA GAA ACT GTG AAG ATC GCT TAT TAT GTC CTATC(SEQ ID NO.3)；
the underlined bases indicate complementary regions where the primer sequence of the DNA probe binds to the RCA template, and the bases in the shaded area indicate the aptamer sequence of the DNA probe.
In yet another embodiment of the present invention, there is provided a method of detecting a histone modification enzyme, the method comprising:
1) incubating the substance to be tested and a histone modification enzyme enzymatic substrate together, and adding a DNA probe for continuous incubation;
2) adding magnetic beads into the step 1) for incubation, washing the magnetic beads after the incubation is finished, dissociating the rest DNA probes, and then continuing high-temperature incubation treatment;
3) adding a rolling circle amplification template into the DNA probe eluted in the step 2) for rolling circle amplification.
Wherein the content of the first and second substances,
in the step 1), the magnetic beads are streptavidin-coated magnetic beads;
the co-incubation treatment conditions were: incubating for 0.5-2 hours at 35-40 ℃, preferably for 40 minutes at 37 ℃;
the conditions for the continuous incubation treatment were: incubating for 0.5-2 hours at 20-30 ℃, preferably incubating for 60 minutes at 37 ℃;
in the step 2), the incubation treatment conditions of adding the magnetic beads are as follows: incubating for 0.5-2 hours at room temperature, preferably incubating for 30 minutes at room temperature;
the high-temperature incubation treatment condition is that incubation is carried out for 0.1-1 hour at 90-100 ℃, and preferably incubation is carried out for 10 minutes at 95 ℃.
In the step 3), the conditions of the rolling circle amplification reaction are specifically as follows: incubating for 0.5-2 hours at 35-40 ℃, preferably for 60 minutes at 37 ℃.
The method for detecting the histone modification enzyme also comprises the step of carrying out fluorescent staining on the rolling circle amplification product in the step 3), thereby realizing real-time fluorescent detection and/or gel electrophoresis and fluorescent spectrum measurement.
In another embodiment of the present invention, there is provided a use of the above-mentioned biosensor and/or detection method for detecting histone modification enzyme activity and/or screening a drug related to histone modification enzyme.
In the application, the application environment for detecting the activity of the histone modification enzyme can be an external natural environment or an in-vivo environment of an organism, including an organism individual, an organ, a tissue or a cell, and can be an organism cell (such as a cancer cell).
The histone modification enzyme related drugs include, but are not limited to, histone modification enzyme inhibitors or histone modification enzyme activators.
The invention is further illustrated by the following examples, which are not to be construed as limiting the invention thereto. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The nucleotide/amino acid sequence information referred to in the examples is shown in the following table:
the underlined bases indicate complementary regions where the primer sequence of the DNA probe binds to the RCA template, and the bases in the darkened portion indicate the aptamer sequence of the DNA probe.
Examples
Cell culture and preparation of cell extracts: human cervical cancer cells (HeLa cells) were cultured in Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum, 50 units per ml penicillin and 50. mu.g per ml streptomycin in a 37 ℃ incubator containing 5% carbon dioxide. Whole cell extracts were prepared using the nuclear extraction kit according to the instructions. The cells were washed twice with 8 ml of pre-cooled phosphate buffer/phosphatase inhibitor buffer (0.8 ml of 10 x phosphate buffer, 6.8 ml of deionized water, 0.4 ml of phosphatase inhibitor) and then removed from the petri dish by gentle scraping of the cells and transferred to a pre-cooled 2 ml tube. The cell suspension was centrifuged at 200 relative centrifugal force for 5 minutes in a 4 ℃ pre-cooled centrifuge. After discarding the supernatant, the cell pellet was resuspended in 300. mu.l lysis buffer (30. mu.l 10 mmol per l dithiothreitol, 267. mu.l lysis buffer, 3.0. mu.l protease inhibitor cocktail). The suspension was incubated on ice for 30 minutes and pipetted up and down for 3 minutes at 2 minute intervals. The suspension was vortexed for 30 seconds and centrifuged at 14000 relative centrifugal force for 20 minutes in a centrifuge pre-cooled to 4 ℃. The supernatant containing the nucleoprotein extract was transferred to a pre-cooled microcentrifuge tube and stored at-80 ℃.
Tip60 detection: 10 microliters of reaction mixture containing the indicated concentration of Tip60, 60 micromoles per liter of polypeptide substrate, 500 micromoles per liter of acetyl-CoA, 1 microliter of reaction buffer (100 millimoles per liter of 4-hydroxyethylpiperazine ethanesulfonic acid, pH 7.5) was incubated at 37 ℃ for 40 minutes. Then 500 nmol/l DNA probe was added and incubated in 1 Xbinding buffer (3 mM potassium chloride, 10 mM disodium hydrogen phosphate, 2 mM potassium dihydrogen phosphate, 10 mM sodium chloride and 5 mM magnesium chloride, pH 7.4) for 60 min at 24 ℃. Subsequently, 20. mu.l of magnetic beads were added and incubated at room temperature for 30 minutes. After washing the beads 3 times, the remaining DNA probes were dissociated with 40. mu.l of deionized water and incubated at 95 ℃ for 10 minutes. For a rolling circle amplification reaction, 50 microliters of the reaction mixture containing 5 microliters of 10 xphi 29 DNA polymerase reaction buffer (500 mmol per liter tris-hcl, 100 mmol per liter magnesium chloride, 100 mmol per liter (ammonium sulfate, 40 mmol per liter dithiothreitol, pH 7.5), 0.4 nmol per liter rolling circle amplification template, 200 mmol per liter dNTP, 0.6U phi29 DNA polymerase, and 40 microliters of eluted DNA probe was incubated at 37 ℃ for 60 minutes, the rolling circle amplification product was stained with 1 × SYBR Gold and measured with a fluorometer, the final volume of fluorescence was measured to be 60 microliters containing 50 microliters of rolling circle amplification product, 6 microliters 10 × br sygold, and 4 microliters deionized water, emission spectra in the range of 520 and 750 nanometers were recorded for data analysis.
Gel electrophoresis: 20 microliters of the final amplification product was analyzed by electrophoresis on a 1% agarose gel (1.4g agarose, 2.8 ml 50 × TAE buffer, 137.2 ml deionized water) at room temperature in 1 × TAE buffer (40 mmol/l Tris-acetic acid, 2 mmol/l EDTA) at 110V constant voltage. A mixture containing 20. mu.l of the rolling circle amplification product, 1 XLoading buffer, 1 XSYBR Gold was loaded onto the gel for electrophoresis, run time 50 min and imaged by an imaging system.
Inhibition of Tip60 activity assay: to evaluate the effect of Tip60 inhibitors, a reaction mixture of 9 microliters containing the indicated concentration of small organic molecule NU9056, 0.54 microliters of 3.71 micromoles per liter Tip60, 0.6 microliters of 1 millimole per liter of polypeptide substrate, 1 microliter of 10 x reaction buffer (1 mole per liter of 4-hydroxyethylpiperazine ethanesulfonic acid, pH 7.5) was incubated at room temperature for 10 minutes. Subsequently, 1 μ l of 5 mmole per liter of acetyl-CoA was added to the mixture and incubated at 37 ℃ for 40 minutes. The Tip60 activity assay followed the same procedure described above.
Experimental principle (as in fig. 1): a carboxy-terminal biotinylated 15 amino acid polypeptide was synthesized as an enzymatic substrate for Tip 60. In addition, a DNA probe consisting of a 39-nucleotide aptamer and a 20-nucleotide primer separated by a spacer for recognition and transduction of the Tip60 enzyme signal into an amplifiable nucleic acid signal was rationally designed. Spacers are introduced to reduce steric hindrance in aptamer-peptide binding and primer-template base pairing. In the presence of the target Tip60, it catalyzes acetylation of the H4 peptide substrate, creating a modification site. Due to the high binding affinity between the modification site and the aptamer, the resulting acetylated peptide substrate can be assembled on streptavidin-coated magnetic beads through specific biotin-streptavidin interactions, thereby forming a magnetic bead-peptide chain-aptamer-primer composite structure. After removing unbound probes by washing the magnetic beads, the probes bound to the modification sites are dissociated under heating at 95 ℃. Due to the perfect complementarity between the primers and the rolling circle amplification template, the released primers of the DNA probes can bind to the rolling circle amplification template, yielding an abundance of single-stranded rolling circle amplification products that can be measured fluorescence simply in a label-free manner by using SYBR Gold dye. Because of the high quantum yield of SYBR Gold dye-nucleic acid complexes, SYBR Gold dyes can bind to nucleic acids to induce signal enhancement by more than 1000-fold. In contrast, in the absence of Tip60, the H4 peptide substrate remained unchanged, did not generate H modification sites, and failed to trigger rolling circle amplification to generate amplified DNA signals. The assay uses aptamers for histone modification enzyme recognition and SYBR Gold dyes for signal measurement without involving any antibody and radioactive or fluorescent labels, and the introduction of histone modification site-specific rolling circle amplification can greatly improve the detection sensitivity.
1. Experimental verification of principle
Products of the rolling circle amplification reaction were analyzed using a 1% agarose gel. As shown in FIG. 2A, in the absence of Tip60 (FIG. 2, A, lane 1), no significant band was observed even in the presence of phi29 DNA polymerase + DNA probe + rolling circle amplification template. In contrast, upon further addition of Tip60, a clear band of the rolling circle amplification product was observed (fig. 2, a, lane 5). Furthermore, in the absence of any of phi29 polymerase (FIG. 2, A, lane 2), DNA probe (FIG. 2, A, lane 3), and rolling circle amplification template (FIG. 2, A, lane 4) in the reaction system, the rolling circle amplification product disappeared, indicating that these components are important for the target-induced rolling circle amplification reaction. These results clearly show that only the presence of the target Tip60 can induce a rolling circle amplification reaction.
Fluorescence measurements were further used to verify target-induced signal amplification. In the absence of Tip60, a lower background signal was observed in the presence of peptide substrate + DNA probe (fig. 2, B, 4), and by further addition of the target Tip60 (fig. 2, B, 3), the signal was enhanced by 83% (result of binding of DNA probe to acetylated peptide substrate). In addition, the fluorescence signal in response to Tip60 (fig. 2, B, 1) was increased by 785% compared to the group without Tip60 (fig. 2, B, 2) due to the addition of Tip60 rolling circle amplification template. The signal amplified by rolling circle amplification was 9.46 times higher than that amplified without rolling circle amplification, indicating that the introduction of amplification facilitates sensitive detection of low abundance histone modification enzymes.
2. Sensitivity test
Under the best experimental conditions, the fluorescence signal of serially diluted Tip60 was measured. The fluorescence signal gradually increased with increasing concentration of Tip60 (fig. 3, a), and a good linear relationship was obtained between the fluorescence intensity at 537 nm and the logarithm of the Tip60 concentration (between 1 picomole per liter and 100 nanomole per liter) (fig. 3, B). The regression equation is that F is 48.5log10C +259.8, linear correlation coefficient 0.9846, where F and C represent fluorescence intensity and Tip60 concentration, respectively. By making an assessmentThe detection limit can be calculated as 220 femtomoles per liter, estimating the mean signal of the blank plus three times the standard deviation. The sensitivity of this assay is 454-fold higher than that of fluorescein isothiocyanate labeled peptide-based fluorometry (100 pmoles per liter), 2224-fold higher than antibody-based and quantum dot-based fluorometry (500 pmoles per liter), and two-fold higher than antibody-based single molecule fluorescence detection (21 pmoles per liter.) the improvement in sensitivity is attributed to the following factors: (1) efficient transduction of Tip60 information to nucleic acid signal by specific aptamer recognition, (2) minimal background due to magnetic separation, and (3) high signal amplification induced by rolling circle amplification.
3. Experiment of specificity
As shown in fig. 4, the specificity of this technique was further investigated by using Bovine Serum Albumin (BSA), DNA adenine methyltransferase (Dam), CpG methyltransferase (CpG), protein kinase a (pka), and T4 polynucleotide kinase (PNK). BSA is a frequently used unrelated protein. Dam and CpG are methyltransferases that catalyze the methylation of adenine residues in the 5' … GATC … 3' sequence and cytosine residues in the 5'. CG.. PKA is responsible for phosphorylation of serine/threonine residues. PNK can catalyze the transfer of phosphate groups from adenosine triphosphate to the 5' -hydroxyl terminus of DNA/RNA. Theoretically, none of these proteins/enzymes can acetylate the peptide chain, so the magnetic beads cannot capture any DNA probes and perform a rolling circle amplification reaction, and no signal is detected. As expected, a high fluorescence signal was observed only in response to the target Tip60, while a low signal similar to the control group was observed in response to BSA, Dam, CpG, PKA, and PNK. These results demonstrate the excellent specificity of the proposed aptamer sensor for Tip 60.
4. Inhibitor assay
For inhibition analysis, small organic molecule NU9056 was used as a model small molecule inhibitor. NU9056 can specifically target Tip60 to reduce Tip 60-catalyzed acetylation of histones. As shown in fig. 5, the relative activity of Tip60 decreased in a dose-dependent manner with increasing concentration of small organic molecule NU 905. The semi-inhibitory concentration value was calculated as 1.7939 micromoles per liter, consistent with the value obtained by the radioactivity measurement (2 micromoles per liter). These results indicate that the assay can be used to screen for Tip60 inhibitors with high selectivity. Has great potential in drug development and disease treatment.
5. Cell experiments
To verify the feasibility of the proposed assay for the actual sample analysis, the endogenous Tip60 activity in the cell extracts of the human cervical cancer cell line (HeLa cells) was measured directly. As shown in fig. 6, a, as the number of cervical cancer cells increased from 0 to 10000 cells, the fluorescence signal increased and even 500 cells could be sensitively detected. Furthermore, 10 micromoles per liter and 100 micromoles per liter of small organic molecule NU9056 can result in a 60.4% and 68.3% reduction in Tip60 activity, respectively (fig. 6, B). These results indicate that this assay can be used for accurate quantification of endogenous Tip60 activity in complex biological samples. These results clearly indicate that the proposed method can be used to detect histone acetyltransferases, providing a new powerful platform for cancer research and non-invasive diagnosis.
It should be noted that the above examples are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the examples given, those skilled in the art can modify the technical solution of the present invention as needed or equivalent substitutions without departing from the spirit and scope of the technical solution of the present invention.
SEQUENCE LISTING
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<211> 80
<212> DNA
<213> template for rolling circle amplification
<400> 3
ctcagctgtg taacaacatg aagattgtag gtcagaactc acctgttaga aactgtgaag 60
atcgcttatt atgtcctatc 80
Claims (10)
1. A biosensor for detecting a histone modifying enzyme, the biosensor comprising at least a histone modifying enzyme enzymatic substrate, a DNA probe, a magnetic bead and a rolling circle amplification template;
wherein the content of the first and second substances,
the histone modification enzyme enzymatic substrate is carboxyl-terminal biotinylated polypeptide;
the DNA probe comprises an aptamer and a primer sequence;
the aptamer recognizes a histone modification enzyme to be detected;
the primer is combined and complemented with the rolling circle amplification template.
2. The biosensor of claim 1, wherein a spacer is disposed between the aptamer and the primer sequence.
3. The biosensor of claim 1, wherein the magnetic beads are streptavidin-coated magnetic beads;
the biosensor further comprises a fluorescent dye comprising a SYBR Gold dye.
4. The biosensor of claim 1, wherein the detection histone modifying enzyme is the acetyltransferase Tip 60.
5. The biosensor of claim 4,
the histone modification enzyme enzymatic substrate is an H4 polypeptide substrate, and the amino acid sequence of the histone modification enzyme enzymatic substrate is as follows: GGKGLGKGGAKRHRK-biotin (SEQ ID NO. 1);
the nucleotide sequence of the DNA probe is as follows: 5'-GTA AGT TAA TTG GAC TTG GTC GTG TGC GGC ACA GCG ATA AAA AAA AAA AAA AAC ACA GCT GAG GAT AGG ACA T-3' (SEQ ID NO. 2);
the nucleotide sequence of the rolling circle amplification template is as follows:
CTC AGC TGT GTA ACA ACA TGA AGA TTG TAG GTC AGA ACT CAC CTG TTA GAA ACT GTG AAG ATC GCT TAT TAT GTC CTA TC(SEQ ID NO.3)。
6. a method of detecting a histone modifying enzyme, the method comprising:
1) incubating the substance to be tested and a histone modification enzyme enzymatic substrate together, and adding a DNA probe for continuous incubation;
2) adding magnetic beads into the step 1) for incubation, washing the magnetic beads after the incubation is finished, dissociating the rest DNA probes, and then continuing high-temperature incubation treatment;
3) adding a rolling circle amplification template into the DNA probe eluted in the step 2) for rolling circle amplification.
7. The method of claim 6, wherein in step 1), the magnetic beads are streptavidin-coated magnetic beads;
the co-incubation treatment conditions were: incubating for 0.5-2 hours at 35-40 ℃, preferably for 40 minutes at 37 ℃;
the conditions for the continuous incubation treatment were: incubating for 0.5-2 hours at 20-30 ℃, preferably incubating for 60 minutes at 37 ℃;
in the step 2), the incubation treatment conditions of adding the magnetic beads are as follows: incubating for 0.5-2 hours at room temperature, preferably incubating for 30 minutes at room temperature;
the high-temperature incubation treatment condition is that incubation is carried out for 0.1-1 hour at 90-100 ℃, and preferably incubation is carried out for 10 minutes at 95 ℃;
in the step 3), the conditions of the rolling circle amplification reaction are specifically as follows: incubating for 0.5-2 hours at 35-40 ℃, preferably for 60 minutes at 37 ℃.
8. The method of claim 6, wherein the method for detecting histone modification enzymes further comprises performing fluorescent staining on the rolling circle amplification product of step 3), thereby performing real-time fluorescence detection and/or gel electrophoresis, and fluorescence spectroscopy.
9. Use of a biosensor according to any one of claims 1 to 5 and/or a detection method according to any one of claims 6 to 8 for detecting histone modification enzyme activity and/or screening for a drug related to a histone modification enzyme.
10. The use according to claim 9, wherein the environment in which the histone modification enzyme activity is detected is an external natural environment or an in vivo environment of an organism, including an organism individual, organ, tissue or cell; or the like, or, alternatively,
the histone modification enzyme related drug comprises a histone modification enzyme inhibitor or a histone modification enzyme activator.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114410793A (en) * | 2022-02-16 | 2022-04-29 | 山东师范大学 | Method for detecting FEN1 activity by label-free fluorescence |
| CN116926169A (en) * | 2022-04-07 | 2023-10-24 | 山东省分析测试中心 | Colorimetric liquid crystal microdroplet biosensor combined with DNA rolling circle amplification technology and sensing system |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111172235A (en) * | 2020-01-15 | 2020-05-19 | 山东师范大学 | Biosensor for detecting cathepsin B and detection method and application thereof |
-
2021
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111172235A (en) * | 2020-01-15 | 2020-05-19 | 山东师范大学 | Biosensor for detecting cathepsin B and detection method and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| SU JIANG 等: "Aptamer-mediated rolling circle amplification for label-free and sensitive detection of histone acetyltransferase activity", 《CHEMICAL COMMUNICATION》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114410793A (en) * | 2022-02-16 | 2022-04-29 | 山东师范大学 | Method for detecting FEN1 activity by label-free fluorescence |
| CN114410793B (en) * | 2022-02-16 | 2024-03-08 | 山东师范大学 | Method for detecting FEN1 activity by label-free fluorescence |
| CN116926169A (en) * | 2022-04-07 | 2023-10-24 | 山东省分析测试中心 | Colorimetric liquid crystal microdroplet biosensor combined with DNA rolling circle amplification technology and sensing system |
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